Infection of humans by Borrelia burgdorferi can result in a multisystemic, chronic disease with diverse manifestations. We are investigation B. burgdorferi at several levels in order to understand some aspects of the disease pathogenesis. These studies include genetic manipulations of the spirochete, characterization of key structural and regulatory elements, and dissemination and clearance of spirochetes in mammalian hosts. 1. Molecular Characterization: Several feature of B. burgdorferi are being addressed. Recombination between the genes encoding the major outer surface proteins (OspA, OspB) of B. burgdorferi can result in both the deletion and generation of osp gene sequences. Such recombinants have been detected and characterized in a number of strains. The molecular basis of additional Osp phenotypes is being investigated. Isolating and studying genes whose expression is increase after a temperature upshift (heat shock genes) will provide information about their roles in cell growth, plasmid replication and adaptation to stress. Understanding the mechanism of linear plasmid replication may yield a new target for antibiotics, in addition to shedding light on ways in which organisms manipulate their genetic material. 2. Gene Transfer: In order to allow genetic analysis of B. burgdorferi, a method for gene transfer into Borrelia is being developed. A derivative of the Borrelia 16 kb linear plasmid will be used as a vector for electroporation, allowing for maintenance of transferred DNA in a native setting. 3. Clearance Studies: Mechanisms that allow B. Burgdorferi to evade host defenses to establish chronic infections in a variety of host tissues remain obscure. During natural infections of mammals, B. burgdorferi must first disseminate from the cutaneous site to other tissues and organs. A mouse clearance model has been developed that makes it possible to quantitatively evaluate the distribution and clearance of intravenously injected B. burgdorferi. It may be possible to correlate patterns of clearance with different structural features of cloned organisms.